This invention relates to biocidal proteins, processes for their manufacture and use, and DNA sequences coding for them. In particular, it relates to antimicrobial proteins isolated from Mirabilis.
Mirabilis comprises about 60 tropical American species, many of which are cultivated for their ornamental value as garden plants. Mirabilis jalapa is commonly known as "four o'clock" or "marvel of Peru", and has white, yellow or red flowers. The tuberous roots of M jalapa are the source of a purgative drug used as a substitute for jalap.
Although plants normally grow on substrates that are extremely rich in fungal organisms, infection remains a rare event. To keep out potential invaders, plants produce a wide array of antifungal compounds, either in a constitutive or an inducible manner. The best studied of these are phytoalexins, secondary metabolites with a broad antimicrobial activity spectrum that are specifically synthesised upon perception of appropriate defence-related signal molecules. The production of phytoalexins depends on the transcriptional activation of a series of genes encoding enzymes of the phytoalexin biosynthetic pathway. During the last decade, however, it has become increasingly clear that some plant proteins can play a more direct role in the control of phytopathogenic fungi. Several classes of proteins with antifungal properties have now been identified, including chitinases, beta-1,3-glucanases, ribosome-inactivating proteins, thionins, chitin-binding lectins and zeamatins.
Researchers at Japan Tobacco Inc have previously extracted an anti-viral protein from Mirabilis jalapa suspension cells (callus initially induced from leaves), and also from root and leaf tissue (Tsutomu Ikeda et al; 1987; Plant Cell Reports, 6, 216-218). This "Mirabilis anti-plant viral protein" (MAP) has a molecular weight of 24 kDa. The amino acid sequence of MAP has been determined and consists of 250 amino acids. A synthetic MAP gene of 759 base pairs has been cloned into a vector and expressed in Escherichia coli (Noriyuki Habuka et al; 1989; Journal of Biological Chemistry, 264 (12), 6629-6637). The following patents have been granted: J88061317 and U.S. Pat. No. 4,701,522 cover the Mirabilis MAP protein extract; J87027797 covers preparations of MAP by culturing callus. The following patent applications have also been filed: J63123386 on MAP obtained by cloning callus cells; J02186988 on preparations of the anti-viral protein by culturing E coli transformants; J01294694 on an anti-viral peptide (NOG-22) from Mirabilis, J01294693 on a similar synthetic peptide (NOG-53), and EP414134 on the gene encoding the anti-viral protein. In addition, Japan Tobacco have filed patent applications covering two anti-viral proteins extracted from Bougainvillea (a closely-related genus in the same family as Mirabilis): BAP-1 has a molecular weight of 33 kDa (J01272598) and BAP-2 has a molecular weight of approximately 30 kDa (J01272599).
We have now purified a new class of potent antimicrobial proteins.